Method for preparation herpes simplex virus subunit vaccine

ABSTRACT

A method for the preparation of an herpes simplex virus subunit vaccine, which comprises subjecting a solution containing glycoproteins gA and gB to column chromatography using, as a gel for chromatography, sulfuric acid ester of cellulose or a crosslinked polysaccharide to adsorb the glycoproteins gA and gB onto the gel in the presence of an anionic surfactant or a nonanionic surfactant, eluting the adsorbed glycoproteins to obtain an eluate containing the glycoproteins, and subjecting the eluate to gel filtration to obtain a filtrate containing the glycoproteins.

The present invention relates to a method for the preparation of aherpes simplex virus subunit vaccine. More particularly, it relates to aherpes simplex virus subunit vaccine which comprises, as an effectivecomponent glycoproteins gA and gB, which are present in both herpessimplex virus type 1 and type 2, said subunit vaccine being useful forprophilaxis of infection by herpes simplex virus type 1 and type 2.

TECHNICAL BACKGROUND AND FIELD OF THE INVENTION

Infection diseases induced by various viruses are almost controlled byvaccination, but the prevention of infection due to herpes simplex virusis still a serious problem. In case of first infection in adult, thesymptoms are usually very severe, and in developed countries, thepopulation having antibodies against herpes simplex virus (hereinafter,referred to as "HSV") is decreasing. This problem will become moreimportant in future. In some countries, it is considered as a kind ofvenereal disease or a neonatal herpes infection.

There are two types of virus in HSV, i.e. type 1 and type 2, and thetype 1 virus infects mainly around lip, and the type 2 virus infectsmainly around genitals.

It is known that both types of viruses are fairly distributed in Japan,and hence, it is important to take effective measures for prophylaxis ofthe virus infection in future.

Most effective prophylactic measures against virus infections areadministration of a vaccine. However, in the case of HSV, development ofvaccine is inhibited because of the specific properties of HSV, i.e.carcinogenicity and latent infection of the virus. It is very difficultto confirm that the infectiousness of HSV is removed in a live vaccineprepared from an attenuated virus or in an inactivated vaccine preparedby inactivating the virus by conventional inactivation processes, suchas addition of inactivating agents or heat-treatment. If a viralinfectiousness is remained in the vaccine, it may induce serious symptomto human body. When such a vaccine is innoculated to human, even thougha symptom does not appear immediately, there is a possibility of latentinfection. Thus, it is very difficult to prove the safety in HSVvaccine. In other aspect, the vaccine to be used for protection to theinfection having low lethal rate such as herpes simplex infection musthighly be purified in order to eliminate undesirable side effect. Fromthese viewpoints, the conventional live vaccine and inactivated vaccineare not practically useful.

PRIOR ART

Under the circumstances, various studies have been done in order todevelop a new vaccine having no danger of HSV infection due tovaccination, among which there are some reports on HSV subunit vaccinewhich might have a possibility of practical use.

It is known that specific glycoproteins of HSV are present in virusenvelope which is surface region of HSV particles and also in the cellmembrane of infected culture cells, and it has been considered that anantibody for preventing HSV infection. Based on the assumption, it hasbeen tried to use, for example, virus envelope components as a vaccinestock.

Skinner et al. have tried to use as a vaccine stock a part of fractionsobtained form kidney cells of brephic hamster (BHK-21) infected with HSVtype 1, which are prepared by destroying the kidney cells by ultrasonictreatment, dissolving the cells by adding thereto 1 v/v % of NonidetP-40 (NP-40, manufactured by Shell Chemical), inactivating the virus bytreating with formalin at 4° C. for 72 hours, and then subjecting themixture to a cushion ultracentrifugation using 20 w/v % sucrose solution[cf. Med. Microbiol. Immunol., 166, 119-132 (1978)].

Kutinova et al. have tried to use as a vaccine stock a supernatantobtained from human embryonic lung cells infected with HSV type 1, whichis prepared by adding 0.5 v/v % Nonidet P-40 to a suspension of thecells and thereby dissolving the cells, and removing nucleus substancesof the cells and nucleocapsid of virus form the dissolved cells bycentrifugation [cf. Arch. Virol., 61, 141-147 (1979)].

Zweerink et al. have reported to use as a vaccine stock a component of aprimary kidney cells of rabbit infected with HSV type 1, which isprepared by dissolving the kidney cells infected with HSV type 1 withTris-EDTA buffer containing 1 v/v % Triton X-100, removing the nucleussubstances of the cells by low speed centrifugation, removing highmolecular weight substances by high speed centrifugation, passing theresulting supernatant through an affinity column packed with Sepharose4B bound with lentil lectin, and eluting the adsorbed components with aneluting solution containing α-methylmannoside and glucose [cf. Infect.Immun., 31, 267-275 (1981)].

Bertland et al. have used as a vaccine stock a virus envelope formchicken embryo fibroblast infected with HSV type 1, which is prepared bydissolving the chicken embryo fibroblast with a phosphate buffercontaining 4 mole of urea, separating the cell components by continuousultracentrifugation, inactivating the virus components contained in thesupernatant by subjecting it to ultrasonic treatment and heat treatmentat 60° C. for 3 hours, decomposing the virus DNA with deoxyribonuclease,by which the virus envelope components is isolated [cf. U.S. Pat. No.4,317,811 (1982)].

Another aspect has been done by Klein et al. [cf. Arch. Virol., 68,73-80 (1981)], that is, a culture supernatant of Vero cells infectedwith HSV type 1 is subjected to continuous ultracentrifugation withsucrose density gradient to obtain purified virus particles, and asuspension of the virus particles is treated with 1 v/v % Triton X-100to destroy the virus particles, and then the resultant is subjected toultracentrifugation with sucrose density gradient to separate into HSVnucleocapsid and virus envelope component, and the latter component isused as a vaccine stock.

Kitces et al. have used as a vaccine stock a virus envelope componenthaving no HSV-originated nucleic acid obtained form human pharyngealcancer epithelial cells (Hep-2 cells) infected with HSV type 1, which isprepared by destroying the epithelial cells with homogenizer,centrifuging the homogenized mixture to separate a supernatant of virusparticle suspension, subjecting the supernatant to inactivation withformalin, adding 1 w/v % of sodium dodecylsulfate and N-lauroylsurcosinesodium salt to the virus particle suspension in order to dissolve them,subjecting the mixture to ultracentrifugation with cesium chloride tocollect a nucleocapsid-free supernatant, treating it withdeoxyribonuclease to give the desired virus envelope having no nucleicacid [cf. Infect. Immun., 16, 955-960 (1977)].

Cappel et al. have tried to use as a vaccine stock a virus envelopecomponent from chicken embryo fibroblast infected with HSV type 1, whichis prepared by subjecting repeatedly the cells to ultrasonic treatmentand freezing-thawing in order to destroy the cells, subjecting theresulting mixture to a low centrifugation and ultrafiltration topartially purify the virus particles, subjecting the crude virusparticles to sugar density gradient ultracentrifugation twice to givepurified virus particles, dissolving the particles with 1 v/v % NonidetP-40, and subjecting the solution to sugar solution cusionultracentrifugation to collect the desired virus envelope component.

The above reports are all concerned with HSV type 1, and as to HSV type2, it is also reported by Hilleman et al. that a subunit vaccine isobtained by subjecting chicken embryo fibroblast infected with HSV type2 to dissolving treatment with Triton X-100, treating the mixture withdeoxyribonuclease, subjecting the mixture to lectin affinitychromatography and Sepharose gel filtration to collect virusglycoproteins and then treating it with aluminum gel a[cf. The HumanHerpes Viruses, 503-509, by Nahmias, A. J. et al., Elsevier, N.Y.(1981)].

In these reports, partially purified virus envelope is used as the virusstock and is treated with aluminum hydroxide gel in order to inceaseimmunogenicity. It has experimentally been confirmed in mice that thevirus envelope is effective as a virus stock, but the process for thepreparation thereof is very complicated in the purification step, andfurther, the purification is not sufficient and hence it is contaminatedwith culture cell components.

It is very important, as mentioned above, that a vaccine for HSV must behighly purified in order to avoid undesirable side effect as low aspossible. It is assumed that the known vaccines are contaminated with afairly amount of proteins from the host, even through the vaccine stockis obtained by extracting it from infected cells or virus particles.Thus, the known vaccines are hardly acceptable as an HSV subunit vaccinefor human in view of less safety.

It is reported by Eberle et al. that anti-sera, which has neutralizingactivity against purified HSV-1 particle against gA and gB are preparedby dissolving treatment human pharyngeal cancer epithelial cellsinfected with HSV with 1 % deocycholic acid and 1 % Tween 40, severaltimes subjecting the mixture to prepartive sodium dodecyl sulfatepolyacrylamide gel electrophresis and sodium dodecylsulfate-hydroxyapatite column chromatography to purify subunit gA andgB.

Eberle et al. also suggested that gA and gB are not the same but theyhave quite similar antigenic determinant sites, and that their bindingrate with glucide are different while their component protein are thesame since anti-gA sera and anti-gB sera react with gB and gArespectively. However, because gA and gB share antigenic determinantsand gA can be changed into gB, it has now been agreed that theglycoprotein gA be designated pgB, a precursor to gB at theInternational Herpes Virus Workshop in Oxford England (1983).

The vaccine stock used for a subunit vaccine against HSV should mostpreferably have antigenic determinants common to HSV type 1 and type 2and the antigenicity is not sufficient by mere producibility ofneutralizing antibody against the virus but should be capable ofcompletely prohibiting the HSV infection. Moreover, as is mentionedabove, the vaccine product should have high safety and excellenteffectiveness and also sufficient storage stability in the form of avaccine preparation. Thus, the practical HSV vaccine product should beprepared from a purified vaccine stock having specific components whichwill be effective against both HSV type 1 and type 2.

It has been found that among the subunit polypeptides present in the HSVenvelope, gA and gB, which are polypeptides common to both HSV type 1and type 2, can exhibit immunological activity sufficient for preventinginfection of the both types of HSV, and the gA and gB can be highlyisolated and purified on an industrial scale by the method of thepresent invention.

An object of the present invention is thus to provide a method forpreparation of improved HSV subunit vaccines against both of HSV type 1and type 2 having high safety, high effectiveness and high stability.

DETAILED EXPLANATION

According to the present invention, highly purified glycoproteins gA andgB can be prepared by subjecting a solution containing such asHSV-specific subunit glycoproteins to affinity chromatography using agel of specific sulfuric acids.

The solution containing such glycoproteins includes a lysate ofmammalian cells infected with HSV (including both of type 1 and type 2,unless otherwise specified), a lysate of partially purified HSVparticles from the culture cells.

The culture cells infected with HSV can express HSV specific subunitglycoproteins on the cell membrane as well as virus particle, and theseculture cells can be used as the starting material in the presentinvention. Besides, there can also be used recombinant culture cellsbeing capable of producing HSV gA and gB which are obtained by geneticengineering technique.

HSV can propagate in wide range of hosts, and the natural host is human,but HSV can also infect to and can be grown in monkey, rabbit, guineapig, hamster, mouse, rat and grown hen's egg. etc. Thus, variousmammalian cells sensitive to HSV can be used in the present invention.For example, brephic hamster kidney cells (BHK-21), Green monkey kidneycells (Vero), human embryonic lung cells (HEL), human pharyngeal cancerepithelial cells (Hep-2), primary rabbit kidney cells (PRK), chickenembryo fibroblast, or the like are usable in the present invention.

For isolating the desired gA and gB, the starting material mayoptionally be subjected to pretreatment. For example, the HSV-infectedculture cells or the culture supernatant is subjected to the treatmentwith a homogenizer or ultrasonic treatment to destroy the cells and thenthe resulting HSV-containing solution is centrifuged to remove crudeinsoluble materials such as cell pieces to give a dispersion of purifiedHSV particles. The thus obtained HSV particles dispersion may also besubjected to the affinity chromatography of the present invention.

The starting materials as above-described are subjected to a dissolvingtreatment with a surfactant. The surfactant used for such dissolvingtreatment includes anion surfactant such as sodium dodecylsulfate (SDS),sodium deoxycholate, and nonionic and nonionic surfactants such asTriton X-100 (tradename of Polyoxy-ethylene ether, manufactured by Rohmand Haas Co.), Nonidet P-40 (tradename of octylphenoxypolyethoxyethanol,manufactured by Shell Company), Tween-20 (tradename of polyoxyethylenesorbitan monolaurate, manufactured by Bio-Rad), but preferably nonionicsurfactants. The addition amount of the surfactant is usually 0.1 to 10v/v %, preferably 0.5 to 2.0 v/v %.

The dissolving treatment is usually carried out by adding a requiredamount of a surfactant to a dispersion of HSV specific subunitglycoproteins and allowing to stand or stirring the mixture at atemperature of 0° to 25° C. for 24 hours.

The glycoproteins extracted by the above dissolving treatment will bere-combined unless any surfactant is present in the system and,according to the present invention the glycoproteins-containing solutionis subjected to affinity chromatography with sulfuric acid ester ofcellulose or a crosslinked polysaccharide, in the presence of an anionicsurfactant or nonionic surfactant, by which the desired gA and gB areisolated.

The sulfuric acid ester of cellulose to be used in the present inventionincludes a sulfuric acid ester of crystalline cellulose or cellulosehaving crystalline area and non-crystalline area. These startingcelluloses are commercially available, for example, as Abicel(manufactured by Asahi Kasei in Japan), Cellulofine GC-15, GH-25,GC-100,or GC-200 (manufactured by Chisso Corp. in Japan).

The sulfuric acid ester of a crosslinked polysaccharide to be used inthe present invention includes a sulfuric acid ester of polysaccharide,such as dextran, cellulose, agarose, which is crosslinked with acrosslinking agent, such as epichlorohydrin, dichlorohydrin,dibromohydrin, ethylene glycol bisepoxypropyl ether. The crosslinkedpolysaccharides are commercially available, for example, as crosslinkeddextran such as Sephadex G-10, G-25, G-50, and G-100 (manufactured byPharmacia in Sweden), crosslinked agarose such as Sepharose C1-2B,C1-4B, and C1-6B (manufactured by Pharmacia in Sweden), and crosslinkedcelluloses such as Cellulofine GCL-25, GCL-90 (manufactured by ChissoCorp. in Japan).

The method of the present invention is based on the fact that the gelsas described above have an affinity to certain polypeptides from HSV.Thus, such gels have a nature of adsorbing the specific subunits, i.e.gA and gB, which are common to HSV-1 and HSV-2, and does not adsorb anyother substances from HSV (e.g. β-lipoprotein). Such gel forchromatography to be used in the present invention is characterized inthat it is prepared by directly sulfating agent such as chlorosulfonicacid or anhydrous sulfuric acid in an organic solvent (e.g. pyridine).Thus, the resultant gel is water-insoluble and highly stable. Further,the gel of the sulfuric acid ester of cellulose or a crosslinkedpolysaccharide exhibits an extremely high adsorbing activity since it isfully sulfated, even at the inner regions thereof. The use of the gel isalso advantageous from an economical standpoint, because it can beeasily prepared at a low cost. The degree of sulfation (content of thesulfonyl group) of crosslinked polysaccharide is usually in the range of0.1 to 40 %, preferably 10 to 40 %, based on the weight of thecrosslinked polysaccharide, and the degree of sulfation of cellulose isusually in the range of 0.1 to 5.0 %, based on the cellulose.

The method of the invention can be carried out in the following manner.Firstly, a sulfuric ester of cellulose or a polysaccharide is packedwithin a column, which is equilibrated with a suitable buffer solutionhaving an ionic strength about 0.001-1.0 and a pH of 5-8, containing0.05-2.0 % of surfactant, which may be selected from the ones used inthe above-mentioned dissolving treatment of HSV-containing materialpreferably 0.1-0.5 %. Such buffer solution may be exemplified by 0.01 Mphosphate buffer solution containing 0.1 M NaCl. After theequilibration, a solution containing HSV-specific subunit polypeptides(gA and gB) prepared by the dissolving treatment, and diluted with thesame buffer solution as used for the equilibration so that not more than100 mg per ml of protein is present, is passed through the column inorder to adsorb the polypeptides onto the gel, followed by washingsufficiently with the same surfactant-containing buffer solution as usedfor the equilibration. Thereafter, the adsorbed polypeptides are elutedfrom the column by passing through the column a suitable buffer solutionhaving an ionic strength larger than that of the surfactant-containingbuffer solution used for the equilibration or the washing, for example,0.01 M phosphate buffer solution containing 0.6 M NaCl and surfactant.The eluate is fractioned, and the fraction containing gA and gB iscollected. From this fraction was prepared a starting material for asubsequent gel filtration, by dialysis of the fraction against a buffersolution which will be used for the gel filtration.

A suitable buffer solution for the gel filtration is the same buffersolution as used for above-mentioned affinity chromatography, i.e., asolution having a pH of 5-8 and an ionic strength about 0.001-1.0,containing 0.05-2.0 %, preferably 0.1-0.5 % of surfactant.

A gel material for the gel filtration in the present invention includesa poly-saccharide or crosslinked polyacrylamide such as cellulose,agarose, or crosslinked dextran, which are commercially available, forexample, as Sephacryl B, Sephadex, Sepharose (manufactured by Pharmaciain Sweden), Bio-Gel P (manufactured by Bio-Rad) Cellulofine(manufactured by Chisso Corp. in Japan) but is not limited to them aslong as they can provide molecular-sieving effects.

By the gel filtration, there are obtained various substances such asthose having molecular weights of about 130,000, about 95,000 and about50,000, among which a fraction having molecular weights of about 90,000to 100,000 us collected as the desired product. This fraction causes theagglutination reaction with anti-gA and -gB monoclonalantibody-sensitized sheep red blood cell, which fact indicates that thefraction consists of gA and gB. In fact, this fraction, when determinedby sodium dodecylsulfate polyacrylamide gel electrophoresis, providesthe substantially the same pattern as gA and gB prepared by affinitychromatography in which there is used a ligand of the monoclonalantibody.

After the gel filtration, the fraction was appropriately condensed,dializing with a buffer solution containing 0.01-0.1 %, preferably0.02-0.05 %, of one of or more nonionic sufactant such as Tween 80,Triton X-100 and so forth.

It is demonstrated that the product thus obtained by the presentinvention contains no impurities when analyzed by electrophresis withSDS-polyacrylamide gel or by immunoblotting method.

A vaccine is usually prepared by adding an immuno adjuvant (e.g.aluminum gel) in order to enhance the antibody producibility whenvaccinated. To the gB-adsorbed aluminum gel suspension thus obtained maybe added a preservative (e.g. thimerosal) in an amount of 0.005 to 0.1w/v % to give the aluminum gel-added vaccine. The gB-adsorbed aluminumgel suspension may be mixed with a stabilizer and optionally apreservative, and then the mixture is lyophilized.

The lyophilized prepartion of HSV subunit vaccine thus obtained can bekept with good storage stability without lowering of antigen titer andfurther can be dissolved rapidly in an injection solution when used.

The present invention is illustrated by the following Preparations andExample, but should not be construed to be limited thereto.

PREPARATION 1

Preparation of crude solution for chromatography.

Vero cells infected with HSV type 1 KOS strain is collected 24 hoursafter the infection, and are dissolved by treating with PBS (pH 7.2-7.4)containing 1 v/v % Triton X-100 at 4° C. for one hour. The dissolvedsolution is centrifuged at 100,000 G for one hour, and then, thesupernatant is collected as a crude glycoproteins-containing solution.

PREPARATION 2

To pyridine (200 ml) is added dropwise chlorosulfonic acid (11 ml) atbelow 0° C. After the addition, the mixture is heated to 65°-70° C. Tothe mixture is added crosslinked agarose (Sepharose CL-6B, manufacturedby Pharmacia) (7.5 g), and the mixture is stirred at 65°-70° C. for 4hours. After the reaction, the reaction mixture is cooled and thenneutralized with aqueous sodium hydroxide. The gel thus obtained isseparated by filtration and washed well with 0.01 M phosphate bufferedsaline solution to give a crosslinked agarose sulfate.

PREPARATION 3

To a mixture (210 ml) of pyridine-chlorosulfonic acid prepared in thesame manner as described in the above Preparation 2 is addedepichlorohydrin-crosslinked dextran (Sephadex G-50, manufactured byPharmacia in Sweden)(7.5 g), and the mixture is reacted at 65°-70° C.for 4 hours. After the reaction, the reaction mixture is cooled andneutralized with aqueous sodium hydroxide. The gel thus obtained isseparated by filtration and washed well with 0.01 M phosphate bufferedsaline solution to give a crosslinked cellulose sulfate (7.2 g).

PREPARATION 4

To a mixture (210 ml) of pyridine-chlorosulfonic acid prepared in thesame manner as described in the above Preparation 3 is added crosslinkedcellulose gel (Cellulofine GLC-25, manufactured by Chisso Corp. inJapan) (30 ml) which has been impregnated with pyridine, and the mixtureis reacted at 65°-70° C. for 4 hours. After the reaction, the mixture iscooled and neutralized with aqueous sodium hydroxide. The gel thusobtained is separated by filtration and washed well with 0.01 Mphosphate buffered saline solution to give a crosslinked agarose sulfate(23 ml).

EXAMPLE

A sulfate gel (200 ml) obtained in the manner as described inPreparation 2-4 is packed within a column and the packed column isequilibrated with 0.01 M phosphate buffered saline solution (pH 7.2)containing 0.1 % Triton X-100 (600 ml). Then, through the column ispassed 100 ml of the supernatant obtained in Preparation 1 (the amountof Protein: about 100 mg/ml), followed by washing with 600 ml of thebuffer solution as used for the equilibration. Thereafter, the adsorbedmaterial is eluted from the column by passing through the column 0.01 Mphosphate buffer solution containing 0.1 % Triton X-100 and 0.6 MNaCl(pH 7.2). The eluate is fractioned, and the fractions are determinedif they contain gA and gB or not by r-PHA method using anti-gA andanti-gB mouse monoclonal antibody sensitized sheep red blood cell, andthen the fraction containing gA and gB is pooled. After the pooledsolution is dialized with 0.01 M phosphate buffered saline (pH 7.2)containing 0.1 % Triton X-100, the solution is condensed with miniconB-15 (manufactured by Amicon) in order to get a sample (4 ml) for gelfiltration.

The Cellulofine GC-700 (manufactured by Chisso Corp in Japan) is packedwithin a column (15 mmo×1,000 mm) and the column is equilibration with0.01 M phosphate buffered saline solution (pH 7.2) containing 0.1 %Triton X-100. Then through the column is passed 1 ml of the sample forthe gel filtration. The gel filtration is carried out in usual manner.Each fraction is determined whether gA and gB are present or not byr-PHA method using anti-gA and anti-gB mouse monoclonal antibody sheepsensitized red blood cell, and the fraction containing gA and gB ispooled.

The fraction gives the same electropholitic pattern as the pure gA andgB vaccine solution obtained with the monoclonal antibody, when analyzedby sodium dodesylsulfate polyacrylamide electropharesis and themolecular weight of the proteins in the fraction is about 95,000. Afterthe dialysis of the pooled fraction with 0.01 M phosphate bufferedsaline containing 0.05 % Triton X-100, there is obtained herpes simplexvirus subunit vaccine stock solution.

The stock solution was added with 0.01 M phosphate buffered salinesolution containing 0.05 % Triton X-100, wherein the amount of proteinis adjusted to a content ration of 50 μg/ml, and after sterilizing thefiltration, the solution is divided and poured into vials (each content:1 ml).

What is claimed is:
 1. A method for the preparation of a herpes simplexvirus subunit vaccine, which comprises subjecting a solution containingglycoproteins gA and gB to column chromatography, using as a gel forchromatography, a sulfuric acid ester of cellulose or a crosslinkedpolysaccharide to adsorb the glycoproteins gA and gB onto the gel in thepresence of an anionic surfactant or nonionic surfactant, said sulfuricacid ester being prepared by treating a gel of cellulose or crosslinkedpolysaccharide with a sulfating agent in an organic solvent, eluting theadsorbed glycoproteins to obtain an eluate containing the glycoproteins,and subjecting the eluate to gel filtration to obtain a filtratecontaining the glycoproteins.
 2. The method as claimed in claim 1,wherein the glycoproteins-containing solution is obtained from culturecells infected with herpes simplex virus subunit vaccine.
 3. The methodas claimed in claim 1, wherein the sulfuric acid ester of crosslinkedpolysaccharide is selected from the group consisting of a crosslinkedcellulose sulfate, a crosslinked agarose sulfate and a crosslinkeddextran sulfate.
 4. The method as claimed in claim 1, wherein thesulfuric acid ester of cellulose is a sulfuric acid ester of crystallinecellulose or a cellulose having a crystalline area and non-crystallinearea.